GSM7711629: Jlat5A8 vRNA AZD Rep2; Homo sapiens; OTHER
To isolate Genomic DNA  for the screen, cells were spun down for each treatment for 5 minutes at 1,000 xg at room temperature, washed in 1x PBS, spun down again, and cell pellets frozen at -80ºC until ready for extraction. Genomic DNA (gDNA) was isolated from each treatment using the QIAamp DNA Blood Midi Kit (Qiagen #51183). Viral RNA (vRNA) was extrated from supernatant of pelleted cells by concentrating over a 20% sucrose solution (70,000 xg for 1 hour at 4C), and then isolating vRNA from pellets using the QIAamp Viral RNA Mini Kit (Qiagen, #52904). Genomic DNA (gDNA) or reverse-transcribed viral RNA (vRNA) were amplified by a two-step PCR (PCR1: 12 cycles, PCR2: 20 cycles) to amplify 20bp sgRNA sequences in the HIV-CRISPR vector. All vRNA was amplified for each sample where appropriate. For gDNA samples, a total of 500-fold coverage of the sgRNAs were amplified (10^6 cells were estimated to contain 6.6μg gDNA) with a maximum of 2μg gDNA per PCR reaction. For each sample, PCR1 reactions were pooled, cleaned up over a QIAquick PCR purification Kit (2 columns per sample), pooled again and used as template for PCR2. For vRNA, all PCR1 was amplified in the second round. For gDNA, 10 uL of pooled PCR1 was used as input for PCR2 (PCR2 primers include Illumina barcodes and adaptor sequences). Amplicons from PCR2 were pooled, purified by double-sided bead cleanup (AMPureXP, Beckman Coulter), quantified with a Qubit dsDNA HS assay and pooled for sequencing.